Conditioned Nictitating Membrane Research Articles

Time course of the rabbit's conditioned nictitating membrane movements during acquisition, extinction, and reacquisition.

The present experiment tested whether or not the time course of a conditioned eyeblink response, particularly its duration, would expand and contract, as the magnitude of the conditioned response (CR) changed massively during acquisition, extinction, and reacquisition. The CR duration remained largely constant throughout the experiment, while CR onset and peak time occurred slightly later during extinction. The results suggest that computational models can account for these results by using two layers of plasticity conforming to the sequence of synapses in the cerebellar pathways that mediate eyeblink conditioning.

Magnitude and timing of conditioned responses in delay and trace classical conditioning of the nictitating membrane response of the rabbit (Oryctolagus cuniculus).

The present experiment characterized conditioned nictitating membrane (NM) movements as a function of CS duration, using the full range of discernible movements (>.06 mm) rather than movements exceeding a conventional criterion (>.50 mm). The CS-US interval was fixed at 500 ms, while across groups, the duration of the CS was 50 ms (trace), 550 ms (delay), or 1050 ms (extended delay). The delay group showed the highest level of acquisition. When tested with the different CS durations, the delay and extended delay groups showed large reductions in their responses when their CS was shortened to 50 ms, but the trace group maintained its response at all durations. Timing of the conditioned movements appeared similar across all manipulations. The results suggest that the CS has both a fine timing function tied to CS onset and a general predictive function tied to CS duration, both of which may be mediated by cerebellar pathways.

Scalar timing varies with response magnitude in classical conditioning of the nictitating membrane response of the rabbit (Oryctolagus cuniculus).

The present experiment was aimed at characterizing the timing of conditioned nictitating membrane (NM) movements as function of the interstimulus interval (ISI) in delay conditioning for rabbits (Oryctolagus cuniculus). Onset latency and peak latency were approximately, but not strictly, scalar for all but the smallest movements (<.10 mm). That is, both the mean and standard deviation of the timing measures increased in proportion to the ISI, but their coefficients of variation (standard deviation/mean) tended to be larger for shorter ISIs. For all ISIs, the absolute timing of the NM movements covaried with magnitude. The smaller movements (approximately, .11-.50 mm) were highly variable, and their peaks tended to occur well after the time of US delivery. The larger movements (>.50 mm) were less variable, and their peaks were better aligned with the time of US delivery. These results are discussed with respect to their implications for current models of timing in eyeblink conditioning.

Evidence From Retractor Bulbi EMG for Linearized Motor Control of Conditioned Nictitating Membrane Responses

Classical conditioning of nictitating membrane (NM) responses in rabbits is a robust model learning system, and experimental evidence indicates that conditioned responses (CRs) are controlled by the cerebellum. It is unknown whether cerebellar control signals deal directly with the complex nonlinearities of the plant (blink-related muscles and peripheral tissues) or whether the plant is linearized to ensure a simple relation between cerebellar neuronal firing and CR profile. To study this question, the retractor bulbi muscle EMG was recorded with implanted electrodes during NM conditioning. Pooled activity in accessory abducens motoneurons was estimated from spike trains extracted from the EMG traces, and its temporal profile was found to have an approximately Gaussian shape with peak amplitude linearly related to CR amplitude. The relation between motoneuron activity and CR profiles was accurately fitted by a first-order linear filter, with each spike input producing an exponentially decaying impulse response with time constant of order 0.1 s. Application of this first-order plant model to CR data from other laboratories suggested that, in these cases also, motoneuron activity had a Gaussian profile, with time-of-peak close to unconditioned stimulus (US) onset and SD proportional to the interval between conditioned stimulus and US onsets. These results suggest that for conditioned NM responses the cerebellum is presented with a simplified "virtual" plant that is a linearized version of the underlying nonlinear biological system. Analysis of a detailed plant model suggests that one method for linearising the plant would be appropriate recruitment of motor units.

Time-specific extinction and recovery of the rabbit's (Oryctolagus cuniculus) conditioned nictitating membrane response using mixed interstimulus intervals.

Extinguishing a conditioned response (CR) has entailed separating the conditioned stimulus (CS) from the unconditioned stimulus (US). This research reveals that elimination of the rabbit nictitating membrane response occurred during continuous CS-US pairings. Initial training contained a mixture of 2 CS-US interstimulus intervals (ISIs), 150 ms and 500 ms. The CRs showed double peaks, one for each ISI. When the 150-ms ISI was removed, its CR peak showed 2 hallmarks of extinction: a decline across sessions and spontaneous recovery between sessions. When a further stage of training was introduced with a distinctive CS using the 150-ms ISI, occasional tests of the original, extinguished CS revealed another hallmark of extinction, specifically, strong recovery of the 150-ms peak. These results support both abstract and cerebellar models of conditioning that encode the CS into a cascade of microstimuli, while challenging theories of extinction that rely on changes in CS processing, US representations, and contextual control.

Recovery of the rabbit's conditioned nictitating membrane response without direct reinforcement after extinction.

Three experiments demonstrated that, following the extinction of an established conditioned stimulus (CSA--e.g., tone), the pairing of a novel, cross-modal stimulus (CSB--e.g., light) with the unconditioned stimulus (US) results in strong recovery of responding to the extinguished CSA. Experiment 1 demonstrated that the recovery of responding to CSA is not the result of US reinstatement but is attributable to pairings of CSB with the US. Experiment 2 demonstrated that the recovery of responding is specific to CSA and is not the result of cross-modal generalization. Experiment 3 revealed that a large number of CSB-US pairings in Stage 1 significantly reduced the amount of recovery to CSA during subsequent CSB-US trials. Experiment 3 also provided unexpected evidence of cross-modal secondary extinction. The extinction and subsequent recovery of responding seen in the present experiments is discussed with respect to possible contributions from contextual associations, CS processing, US processing, conditioned response expression, and layered excitatory associations.

Hippocampal Theta Oscillations and Classical Conditioning

Studies are reviewed that support a hypothesized role for hippocampal theta oscillations in the neural plasticity underlying behavioral learning. Begun in Richard F. Thompson's laboratory in the 1970s, these experiments have documented a relationship between free-running 3- to 7-Hz hippocampal slow waves (theta) and rates of acquisition in rabbit classical nictitating membrane (NM) conditioning. Lesion and drug manipulations of septohippocampal projections have affected NM and jaw movement conditioning in ways consistent with a theta-related brain state being an important modulator of behavioral acquisition. These findings provide essential empirical support for the recently developed neurobiological and computational models that posit an important role for rhythmic oscillations (such as theta) in cellular plasticity and behavioral learning.

Acquisition of Eyeblink Conditioning Is Critically Dependent on Normal Function in Cerebellar Cortical Lobule HVI

Classical conditioning of the nictitating membrane response (NMR)/eyeblink response of rabbits is a simple form of cerebellar-dependent, associative motor learning. Reversible inactivations of the cerebellar nuclei and inferior olive have implicated the olivo-cortico-nuclear loop in the acquisition of nictitating membrane conditioning, but the role of the cerebellar cortex in acquisition has not been tested directly. Here we have used local infusions of the water-soluble, disodium salt of 6-cyano-7-nitroquinoxaline-2,3-dione reversibly to block cerebellar cortical AMPA/kainate receptors in lobule HVI during acquisition training. After the drug effects dissipated, there was no evidence that acquisition had taken place; the subjects behaved as if naive. Further training without inactivation then allowed normal acquisition, and further inactivations during performance of conditioned responses abolished these established responses. There was a strong correlation between the inactivation effects on acquisition and subsequent inactivation effects on performance, indicating that the same eyeblink-control cortical microzones are engaged in learning and expressing this behavior. The cortical component of the olivo-cortico-nuclear loop is essential for acquisition of classically conditioned nictitating membrane response learning, and eyeblink control areas in HVI are critical. Our findings are consistent with models of cerebellar learning that assign essential plasticity to the cortex or to a distribution between levels in olivo-cortico-nuclear modules.

The effect of brachium conjunctivum transection on a conditioned limb response in the cat

Seven cats were trained to perform a forelimb conditioned response to a paired tone conditioned stimulus (CS)/shock unconditioned stimulus (UCS). Brachium conjunctivum section ipsilateral to the trained limb was carried out following criterion conditioned response (CR) performance. Lesion sites were identified histologically and further confirmed by observation of cellular changes in the dentate and interpositus nuclei ipsilateral to the section. The brachium conjunctivum was found to have been sectioned in four of the seven subjects. Each of these animals demonstrated a total or near-total loss of the CR. Extended postoperative training resulted in no increase in CR performance levels. The unconditioned response (UCR) remained unaffected, as did limb placing, accuracy of striking at moving objects, grooming, running and walking. The results are discussed in the context of an earlier report by McCormick et al. [Bull Psychonom Soc 1981;18:103–5], in which section of the superior cerebellar peduncle was found to abolish a conditioned nictitating membrane response in the rabbit. Taken together, they support the contention of Lavond [Annu Rev Psychol 1993;44:317–42], Thompson [In: Sprague JM, Epstein AN, editors. Progress in Psychobiology and Physiological Psychology. New York: Academic Press 1983, pp. 167–96], Yeo et al. [Behav Brain Res 1984;13:261–66; Exp Brain Res 1985;60:87–98; Exp Brain Res 1985;60:99–113; Exp Brain Res 1992;88:623–38.] and others that the cerebellum represents a critical site for acquisition and retention of a conditioned memory trace.

The effect of feature-target intervals in conditional discriminations on acquisition and expression of conditioned nictitating membrane and heart rate responses in the rabbit

Nictitating membrane (NM) and heart rate (HR) responses were investigated in a conditional discrimination (A→X+ vs. B→X−), using feature-target intervals of 0, 5, 15, and 45 sec. Conditional control of NM responses, but not of HR responses, was acquired to the 400-msec X stimulus in all the groups tested. However, differential conditioning of both the NM and the HR responses to A versus B feature cues appeared for the three shorter intervals. Following acquisition, all the rabbits were tested with the four different feature-target intervals. All the groups showed a gradient of NM responding to X, in which the highest level of responding occurred at or near the interval used in training. The results are discussed with respect to the relationship of simple conditioning of the feature cues to their control over responding during presentation of the target stimulus, the putative role of HR as an index of preparatory processes during presentation of feature cues, and mechanisms of temporal specificity in conditional discriminations.

Cerebellar Cortical AMPA–Kainate Receptor Blockade Prevents Performance of Classically Conditioned Nictitating Membrane Responses

Classical conditioning of the nictitating membrane-eye blink response of rabbits is a simple form of associative motor learning. Lesion studies have shown that performance of learned responses is dependent on the cerebellum, but they have not shown whether there is storage of memories within the cerebellum or distinguished the roles of the cerebellar cortex and nuclei. Reversible inactivations of the cerebellar nuclei have directly implicated the cerebellum in the acquisition of nictitating membrane conditioning, but previously the cerebellar cortex has not been reversibly inactivated to assess its contribution to the performance or acquisition of conditioned responses. Here we use the water-soluble disodium salt of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) reversibly to block cerebellar cortical AMPA-kainate receptors in lobule HVI and quantitative autoradiography to map its distribution. Conditioned responses are completely, but reversibly, abolished for 10-60 min depending on the concentration of the CNQX infusion and its location within HVI. Zebrin immunohistochemistry was used to define the optimal cortical infusion site that, we suggest, corresponds to the location of the eye blink control regions. We confirm that areas in HVI are essential for the expression of classically conditioned nictitating membrane responses, and we establish a method to analyze the role of cerebellar cortex in the acquisition of this form of motor learning.

Acute inactivation of the inferior olive blocks associative learning.

Can acute inactivation of the inferior olive block associative learning? We anaesthetized the inferior olive with lidocaine while rabbits simultaneously: (i) performed conditioned nictitating membrane responses to a flashing light to which they had already been trained; and (ii) underwent their first experience with classical conditioning of the same response to a tone. Inactivation of the inferior olive immediately and reversibly abolished the performance of conditioned responses and prevented learning during rabbits' initial conditioning with a tone-conditioned stimulus. When olivary function was restored, rabbits showed no signs of having learned under olivary anaesthesia. The experiment demonstrates that an acute disruption in olivary function can block learning, in addition to severely degrading motor control. The results are interpreted to indicate the importance of the inferior olive in optimizing learning, perhaps through a general role in regulating temporal processing.

Acquisition of a new-latency conditioned nictitating membrane response--major, but not complete, dependence on the ipsilateral cerebellum.

Classical conditioning of the nictitating membrane response (NMR) of rabbits is simple associative learning of a motor response. In several two-stage experiments, reversible inactivations of the deep cerebellar nuclei in stage 1 appeared to prevent acquisition of NMR conditioning in naive rabbits--no conditioned responses (CRs) were evident after inactivations were lifted in stage 2. Results of a three-stage experiment were different. When subjects were first trained with a light conditional stimulus (CS) in stage 1, reversible cerebellar inactivations during conditioning to a different, tone CS during stage 2 did not appear to prevent new learning because CRs to the tone CS were evident when the inactivation was lifted. Results from the two-stage experiments support the suggestion that the cerebellum is essential for the acquisition of NMR conditioning, but results from the three-stage experiment do not. Here, we use a three-stage design with different interstimulus intervals (ISIs) in stages 1 and 2. Because CRs develop with latencies-to-peak dependent on the ISI, learning during stage 1 can be dissociated from that accruing in stage 2. Complete inactivation of the ipsilateral cerebellar nuclei with muscimol substantially but not completely prevented learning with the second ISI during stage 2 because small CR peaks around the stage 2 ISI could be detected in some subjects after the inactivation had been lifted in stage 3. We suggest that the weak levels of conditioning possible during unilateral inactivation depend on the contralateral cerebellum or on extracerebellar circuitry and that these may be capable of supporting transfer of conditioning in a previous three-stage experiment. But, we confirm that normal NMR conditioning is critically dependent on the ipsilateral cerebellum.

Neuronal Activity in the Medial Prefrontal Cortex during Pavlovian Eyeblink and Nictitating Membrane Conditioning

The present study assessed Pavlovian eyeblink (EB) conditioning, using tones and periorbital shock as the conditioned and unconditioned stimuli (CS and US), and nictitating membrane (NM) conditioning, using tones and airpuffs as the CS and US. During each experiment, CS-evoked changes in multiple-unit activity (MUA) in the medial prefrontal cortex (mPFC) were recorded. Concomitant heart rate (HR) conditioned responses (CRs) were also recorded. A nonassociative control group received explicitly unpaired presentations of the CS and US in each experiment. Increases in both NM and EB CRs occurred over sessions in the paired, but not the unpaired, groups. Decelerative HR CRs also occurred in the eyeshock, but not the airpuff, group. Although tone-evoked increases in neuronal activity were obtained during 10 initial tone-alone presentations in all groups, this activity habituated over trials. CS-evoked increases in neuronal activity also occurred, but this activity was considerably greater in the group that received periorbital shock as the US. During subsequent extinction trials, decreases in tone-evoked neuronal activity occurred in this group, compared with the previous CS/US paired trials. CS-evoked MUA increases were minimal during all except the pretraining phase of the study in the CS/US unpaired control groups and in the paired airpuff group. These findings show that neuronal activity during associative learning occurs in the mPFC during Pavlovian EB, as well as HR conditioning, but this activity apparently reflects an affective component to learning that is only indirectly related to skeletal conditioning.

Reversible inactivations of the cerebellum prevent the extinction of conditioned nictitating membrane responses in rabbits.

1. Studies show that reversible inactivation of the anterior interpositus nucleus (AIP) of the cerebellum with muscimol (a GABAA agonist) prevents acquisition of the classically conditioned nictitating membrane response (NMR) in the rabbit. Here, we have used reversible inactivations of the AIP with muscimol to investigate the role of the cerebellum in the extinction of this response. 2. Experimental subjects were implanted with cannulae targeted to the AIP, through which muscimol could be infused via an injector cannula. This experiment was divided into three phases lasting 4 days, separated by 3 day intervals. Experimental and unoperated control subjects received acquisition training in phase 1; in phases 2 and 3 they received extinction training. 3. Presentation of the conditioned stimulus (CS) alone in phase 2 produced normal extinction in control subjects. Muscimol inactivation of the AIP in experimental subjects during phase 2 prevented extinction of conditioned responses (CRs), shown by initial high CR frequency in the first post-drug session of phase 3, which then extinguished in a manner indistinguishable from controls in phase 2. 4. Our findings support the suggestion that similar cerebellar circuitry is engaged in acquisition and extinction of NMR conditioning.

Reversible inactivations of the cerebellum with muscimol prevent the acquisition and extinction of conditioned nictitating membrane responses in the rabbit.

Lesions of the cerebellum severely impair the classically conditioned nictitating membrane response (NMR) in rabbits. Thus, the cerebellum is essential for the production of conditioned responses (CRs), either because it is actively involved in NMR conditioning or because damage to it causes motor or other general deficits. To distinguish between these alternatives, the cerebellum may be inactivated during training. Inactivation of the cerebellum during acquisition training might result in the absence of CRs on initial trials of subsequent training without the neuronal blockade. The blockade may have prevented learning but it may have produced other deficits that require time or further training to overcome. This problem can be addressed by inactivating the cerebellum during extinction training. If inactivation during extinction training results in the immediate production of CRs when training is resumed without the blockade, then it may be concluded that extinction learning was prevented by the blockade-the presence of CRs argues against any deficits not associated with learning. We used muscimol to inactivate the cerebellum and test its involvement in acquisition and extinction of NMR conditioning in the same subjects. We injected muscimol close to the interpositus nucleus of the cerebellum 1 h before each of four daily training sessions of delay conditioning. Almost no CRs were produced in these training sessions-there was little or no acquisition of NMR conditioning during cerebellar inactivation. The subjects were then trained for four daily sessions without injections of muscimol. There were no CRs on initial trials of the first session of retraining, but all subjects produced CRs by the end of this session. The subjects then received four daily sessions of extinction training with muscimol inactivation of the nuclei-no CRs were produced. Extinction training then continued for four daily sessions without muscimol inactivation. On the first of these sessions, all subjects immediately produced high levels of CRs. These responses then extinguished within and between sessions with characteristic beginning-of-session spontaneous recovery. There was little or no extinction of NMR conditioning during cerebellar inactivation. After inactivation, the muscimol- inactivated subjects went on to acquire and extinguish NM responses at rates similar to those of appropriate controls. We conclude that cerebellar circuitry is essential for, and actively engaged in, both acquisition and extinction of this simple form of motor learning.

A neural model of timed response learning in the cerebellum

A spectral timing model is developed to explain how the cerebellum learns adaptively timed responses during the rabbit's conditioned nictitating membrane response (NMR). The model posits two learning sites that respectively enable conditioned excitation and timed disinhibition of the response. Long-term potentiation of mossy fiber pathways projecting to interpositus nucleus cells allows conditioned excitation of the response's adaptive gain. Long-term depression of parallel fiber-Purkinje cell synapses in the cerebellar cortex allows learning of an adaptively timed reduction in Purkinje cell inhibition of the same nuclear cells. A spectrum of partially timed responses summate to generate an accurately timed population response. In agreement with physiological data, the model Purkinje cell activity decreases in the interval following the onset of the conditioned stimulus, and nuclear cell responses match conditioned response (CR) topography. The model reproduces key behavioral features of the NMR. including the properties that CR peak amplitude occurs at the unconditioned stimulus (US) onset: a discrete CR peak shift occurs with a change in interstimulus interval (ISI) between conditioned stimulus (CS) and US; mixed training at two different ISIs produces a double-peaked CR; CR acquisition and rate of responding depend unimodally on the ISI; CR onset latency decreases during training; and maladaptively timed, small-amplitude CRs result from ablation of cerebellar cortex.

Long-Term Memory and Extinction of the Classically Conditioned Rabbit Nictitating Membrane Response

The purpose of the present experiment was to examine the duration of long-term memory for classical conditioning of the rabbit nictitating membrane response (NMR). Anecdotal accounts of memory for the rabbit NMR suggest that a conditioned nictitating membrane response (CR) may occur as long as a year after acquisition. The data from the present between-subjects experiment indicate that CRs (1) are robust if tested after 1 month, (2) still occur if tested after 3 months, and (3) are initially absent if tested after 6 or 9 months but then reemerge over the course of extinction. The data also indicate that reacquisition of CRs 6 or even 9 months after training is significantly more rapid than original acquisition and thus represents a substantial level of savings. These results provide the first systematic, empirical assessment of the long-term memory capacity of the rabbit for classical conditioning of the NMR and suggest that the rabbit NMR preparation is a model suitable for studying the behavioral and physiological processes involved in long-term memory, retention, and forgetting.

Recoverable and nonrecoverable deficits in conditioned responses after cerebellar cortical lesions

This study reexamined the effects of unilateral damage to cerebellar hemispheral lobule VI on the rabbit's conditioned nictitating membrane (NM) response. Extensive unilateral removal of hemispheral lobule VI in 11 rabbits impaired ipsilateral conditioned responses as reflected by reductions of 52% in mean frequency and 53% in mean amplitude during test trials on the first postoperative session. The decreases in the amplitude and frequency of conditioned responses were highly correlated (r = 0.82). The frequency of conditioned responses recovered to control levels but their amplitudes remained reduced such that the correlation between these two measures of responding was no longer significant by the 12th postoperative conditioning session. The decrease in the amplitude of conditioned responses was not accompanied by changes in onset latency or rise time. There was no significant impairment of conditioned responses in surgical controls and animals with only partial damage to hemispheral lobule VI. It was concluded that hemispheral lobule VI plays an important role in the regulation of motor centers in the brainstem so as to facilitate the initiation and optimum execution of the conditioned NM reflex. This cortical regulation of the conditioned NM response may contain learned elements; however, these cannot be resolved with lesion methods, nor has their existence been proven in this or other lesion studies. Nevertheless, the results of this study do demonstrate that the cerebellar cortex cannot be considered as the single locus necessary for NM conditioning.

Posttraining effects of amphetamine, chlorpromazine, ketamine, and scopolamine on the acquisition and extinction of the rabbit's conditioned nictitating membrane response.

Posttraining effects of amphetamine, chlorpromazine, ketamine, and scopolamine on the acquisition and extinction of the rabbit's conditioned nictitating membrane response.